Cell-Based Composite Materials with Programmed Structures and Functions

ABSTRACT

The present invention is directed to the use of silicic acid to transform biological materials, including cellular architecture into inorganic materials to provide biocomposites (nanomaterials) with stabilized structure and function. In the present invention, there has been discovered a means to stabilize the structure and function of biological materials, including cells, biomolecules, peptides, proteins (especially including enzymes), lipids, lipid vesicles, polysaccharides, cytoskeletal filaments, tissue and organs with silicic acid such that these materials may be used as biocomposites. In many instances, these materials retain their original biological activity and may be used in harsh conditions which would otherwise destroy the integrity of the biological material. In certain instances, these biomaterials may be storage stable for long periods of time and reconstituted after storage to return the biological material back to its original form. In addition, by exposing an entire cell to form CSCs, the CSCs may function to provide a unique system to study enzymes or a cascade of enzymes which are otherwise unavailable.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/638,315, filed Apr. 25, 2012, which is incorporated herein byreference in its entirety. This application is also a divisional ofprior application Ser. No. 13/869,799, filed Apr. 24, 2013, which isalso incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under contract no.DE-AC04-94AL85000 awarded by the U. S. Department of Energy to SandiaCorporation. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to cell-based composite materials and, inparticular, to cell-based composite materials with programmed structuresand functions.

BACKGROUND OF THE INVENTION

The synthesis of inorganic materials with controlled and complex formshas been facilitated through discoveries such as vesicle, micelle andliquid crystalline templating of silicates which provided inspiration toexplore a range of templating strategies based on self-assembledmolecular precursors, colloids, and biological templates and vessels.See C. T. Kresge et al., Nature 359, 710 (1992); J. S. Beck et al., J AmChem Soc 114, 10834 (1992); S. Mann and G. A. Ozin, Nature 382, 313(1996); C. J. Brinker et al., Adv Mater 11, 579 (1999); E. Pouget etal., Nat Mater 6, 434 (2007); C. L. Chen and N. L. Rosi, Angew Chem IntEd 49, 1924 (2010); K. E. Shopsowitz et al., Nature 468, 422 (2010); C.Boissiere et al., Adv Mater 23, 599 (2011); B. T. Holland et al.,Science 281, 538 (1988); B. Hatton et al., Proc Natl Acad Sci USA 107,10354 (2010); A. Stein et al., Chem Mater 20, 649 (2008); O. Paris etal., MRS Bull 35, 219 (2010); D. Van Opdenbosch et al., J Mater Chem 26,1193 (2011); and K. J. C. van Bommel et al., Angew Chem Int Ed 42(9),980 (2003). A driving force for these efforts is the many complexinorganic structures found in nature. An oft-cited example is thehierarchical composites built by silica condensing microorganisms suchas diatoms, which have generated substantial scientific interest forover a century. See P. Fratzl and S. Weiner et al., Adv Mater 22, 4547(2010). Diatoms display complex 3D architectures with great structuralcontrol over nano- to millimeter length scales. However, despite somesuccess toward elucidating mechanisms of diatom biomineralization, thein vitro synthesis of 3D diatom-like forms has remained elusive. Diatomsilica has found numerous applications including as a chemicalstabilizer, absorbent, filter medium, and fine abrasive, and the lack ofsynthetic analogues has facilitated recent investigations to employdiatom frustules as starting materials for shape-preserving chemicaltransformations into functional nanomaterials. See K. H. Sandhage etal., Handbook of Biomineralization: Biomimetic and bioinspiredchemistry, 235 (2007); D. Losic et al., Adv Mater 21, 2947 (2009); andZ. Bao et al., Nature 446, 172 (2007). Given the potential of thisbiosilica, it would be desirable to be able to wield control over thesilica structure in order to achieve broader applicability; however,strategies to direct diatom morphology using chemical and geneticapproaches has proven challenging. See M. Hildebrand, J NanosciNanotechno 5, 146 (2005); H. E. Townley et al., Nanotechnology 18,295101 (2007); and N. Kroger, Curr Opin Chem Biol 11, 662 (2007).Therefore, an ability to generate cell frustules from more malleabletemplates such as mammalian cells would provide greater access tonatural and engineered cell heterogeneity—both structure and function—tobe exploited in the design of complex materials.

To these ends, biomineralization by silica condensing microorganismsoffers key lessons. The discovery of biogenic peptides that catalyzesilica condensation subsequently has motivated the extensiveinvestigation of the interaction of natural and synthetically-derivedpeptides and proteins with silica and its precursors. See N. Kroger etal., Proc Natl Acad Sci USA 97, 14133 (2000); N. Kroger et al., Science286, 1129 (1999); J. N. Cha et al., Proc Natl Acad Sci USA 96, 361(1999); E. Pouget et al., Nat Mater 6, 434 (2007); N. Kroger et al.,Science 298, 584 (2002); T. Coradin et al., Colloids Surf B 29, 189(2003); A. Bassindale et al., J Mater Chem 19, 7606 (2009); C. Gautieret al., Colloids Surf B 65, 140 (2008); M. Dickerson et al., Chem Rev108, 4935 (2008); and S. V. Patwardhan et al., Chem Commun 9, 1113(2005). Identification of silica associated biomolecules such aslong-chain polyamines and the silaffin peptides has led to a generalunderstanding of the tenets by which macromolecules controlpolymerization of silica precursors into silica assemblies. See N.Kroger et al., Proc Natl Acad Sci USA 97, 14133 (2000); N. Kroger etal., Science 286, 1129 (1999); and M. Hildebrand, Chem Rev 108, 4855(2008). However, silica morphogenesis at the meso- and micro-scales mustinvolve both transport of soluble silica precursors and their directeddeposition by biomolecular templating or structural elements. See B.Tesson and M. Hildebrand, PloS one 5, e14300 (2010); E. Brunner et al.,Angew Chem Int Ed 48, 9724 (2009); and A. Scheffel et al., Proc NatlAcad Sci USA 108, 3175 (2011). Likely, these larger scale molecularassemblies direct the assembly of silica building blocks, formed in thesilica deposition vesicle (SDV), into complex structures.

Therefore, Khripin et al. recently examined whether synthetic 3D proteinscaffolds could direct/template silica deposition provided theappropriate silica precursors and chemical conditions. See C. Y. Khripinet al., ACS Nano 5, 1401 (2011). They showed that microfabricatedprotein hydrogels could template silica volumetrically into mechanicallystable, nano- to micro-scale biocomposites with user-defined 3D featuresidentical in size and shape to those of the template.

These features were preserved following removal of the organic componentto form a porous silica replica. Importantly, proteins of diverseproperties (e.g., isoelectric point; pI) directed silica condensationunder identical solution conditions (100 mM silicic acid, pH 3), whichis somewhat contrary to the generally held understanding that cationicspecies (e.g., proteins with pI>7) are required for biogenic silicadeposition. See A. Bassindale et al., J Mater Chem 19, 7606 (2009).These protein hydrogels are highly concentrated (>40% protein by wtvol⁻¹), producing a locally crowded 3D molecular environment, which actsto capture and concentrate silica precursors (mono-, oligo-silicic acid,and nanoparticles) via hydrogen bonding and other non-covalentinteractions, promoting their further condensation and conversion tocovalently bonded siloxane replicas.

However, a need remains for a method of directed silica condensation innaturally crowded molecular environments, such as cells, under similarconditions.

SUMMARY OF THE INVENTION

The present invention is directed to the use of silicic acid totransform biological materials, including cellular architecture intoinorganic materials to provide biocomposites (nanomaterials) withstabilized structure and function. In the present invention, there hasbeen discovered a means to stabilize the structure and function ofbiological materials, including cells, biomolecules, peptides, proteins(especially including enzymes), lipids, lipid vesicles, polysaccharides,cytoskeletal filaments, tissue and organs with silicic acid such thatthese materials may be used as biocomposites. In many instances, thesematerials retain their original biological activity and may be used inharsh conditions which would otherwise destroy the integrity of thebiological material.

In certain instances, these biomaterials may be storage stable for longperiods of time and reconstituted after storage to return the biologicalmaterial back to its original form. In addition, by exposing an entirecell to form CSCs, the CSCs may function to provide a unique system tostudy enzymes or a cascade of enzymes which are otherwise unavailable.

The present invention is more particularly directed to a method tosynthesize cell/silica composites, comprising incubating a plurality ofcells in a silicic acid solution to provide cell/silica compositeparticles. The plurality of cells can comprise mammalian cells,prokaryotic cells, plant cells, cultured cells, or even tissue or othermulticellular organisms. The plurality of cells can be genetically,chemically, or physically modified. The method can further comprisefixing the plurality of cells in a fixative prior to the incubatingstep. The silicic acid solution is preferably osmotically balanced(isotonic). The silicification can be performed in a suspension and theresulting cell/silica composite particles can be dehydrated to providemonodisperse cell/silica composite particles. Alternatively, the cellscan be plated or patterned on a substrate prior to incubation. Themethod can further comprise calcinating the cell/silica compositeparticles at an elevated temperature to provide silica replicas. Thesilicified cells can be used for the replication of biological surfaces.For example, the method can further comprise reconstituting a cellularfunction on the silica replicas, such as by introducing an amphiphiliclipid bilayer on the outer surface of the silica replicas and by theaddition of biological material (e.g., enzymes, ATP, organelles, RNA,etc.). Therefore, the silicified cells can provide a substrate for 3Dcell culture, cell interaction and differentiation, or tissueregeneration. The substrate can be de-silicified to enable stable,long-term preservation of biological material. The method can furthercomprise pyrolyzing the cell-silica composite particles at a hightemperature in an inert atmosphere to provide carbonized-cell/silicacomposite particles. The carbonized particles can be electricallyconductive or provide a conductive support. For example, thecarbonized-cell/silica composite particles provide a biocatalyst. Forexample, the cell/silica composites, silica replicas, orcarbonized-cell/silica composite particles can provide a separationmedium, adsorbent, or absorbent.

Tissue-derived cultured cells exhibit a remarkable range ofmorphological features in vitro, depending on phenotypic expression andenvironmental interactions. Translation of these cellular architecturesinto inorganic materials according to the present invention provides newroutes to generate hierarchical nanomaterials with stabilized structuresand functions. As an example, the fabrication of cell/silica composites(CSCs) and their conversion to silica replicas using mammalian cells asscaffolds to direct complex structure formation is described herein.Under mildly acidic solution conditions, silica deposition is restrictedto the molecularly crowded cellular template. Inter- and intracellularheterogeneity from the nano- to macro-scale is captured anddimensionally preserved in CSCs following drying and subjection toextreme temperatures allowing, for instance, size and shape preservingpyrolysis of cellular architectures to form conductive carbon replicas.The structural and behavioral malleability of the starting material(cultured cells) provides opportunities to develop robust and economicalbiocomposites with programmed structures and functions.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form part ofthe specification, illustrate the present invention and, together withthe description, describe the invention. In the drawings, like elementsare referred to by like numbers.

FIG. 1 shows silicification of mammalian cells cultured on flatsubstrates. Provided are (a) a schematic describing the process of cellsilicification; and (b) an optical image field of AsPC-1 cellsthroughout the steps shown in (a) of silicification. Images in panels 1and 2 show hydrated cells and panels 3 and 4 show dehydrated compositesand silica replicas. Insets show representative EDS spectra of cells atthe various stages. Provided are (c) a close-up differentialinterference contrast (DIC) image of the cell noted by the white arrowin b, rightmost panel; and (d) a representative SEM image of AsPC-1templated cell silica following calcination. Scale bars: 40 μm (b); 5 μm(c and d).

FIG. 2 shows CSC particles derived from cell suspensions. Provided are(a) a schematic illustration the formation of CSC particles; and (b) animage of a dry powder comprised of monodisperse CSC particles resultingfrom silicification, as for adherent cells, and dehydration. The largepanel shows a close-up SEM of a 4T1 templated CSC displaying a ruffledexternal surface. Provided are (c) close-up SEMs of CSC particlesderived from a variety of tissues. Insets show the whole particle.Provided are (d) calcined CSCs templated from human erythrocytes showingnormal to increasingly abnormal/crenate morphology resulting fromincreasing levels of osmotic stress (left to right). Also provided are(e) calcined CSCs derived from RBL-2H3 cells before (left panels) andafter (right panels) IgE crosslinking. Scale bars for (c)-(e): 1 μm.

FIG. 3 are SEM images of clusters of calcined (500° C., 3 hrs) silicareplicas templated from 4T1 cells incubated for 0, 30 and 150 minutes in5 μm doxorubicin to induce apoptosis. Scale bars: 2 μm.

FIG. 4 are SEM images. Provided are (a) an SEM of AsPC-1 templated CSCfeatures (indicated by (+) silicic acid); and (b) an SEM of cells fixedand dehydrated using standard procedures (indicated by (−) silicicacid). Magnified features are indicated by arrows. Also provided are (c)SEM images of SK-OV-3 suspension cultured cells dried against asubstrate with (+) and without (−) silicic acid treatment.

FIG. 5 shows SEM images of calcination of fixed ASPC1 cells in theabsence of silicic acid treatment.

FIG. 6 shows an SEM analysis of filopodia mean width of fixed cells (75nm), cell/silica composites (86 nm), and silica (79 nm) derived fromsubstrate-bound differentiated AsPC-1 cells. Error bars indicate thestandard error of the mean.

FIG. 7 shows cross-sectional SEM imaging of CSCs enabled by a simplefracture technique. Provided is (a) an SEM image of the fracture of CSCson a coverslip provides clean sectioning to reveal intracellularfeatures using SEM. The right panel is a close up view of the sectionedcell. Arrows indicate nuclear pore complexes. Provided are (b) an SEMsection of a CSC showing multilayer, endoplasmic reticulum-likestructures, indicated by arrows; (c) an SEM of a calcined CSC sectionedon glass showing a 30 nm membrane templated silica structure; and (d) anSEM of Filopodia-templated upright protrusions that (1) are encased in asmooth silica membrane (2) overlying roughened, particle-based features(3) in a calcined and sectioned CSC. Arrows in (b)-(d) insets point tothe area of magnification. All scale bars: 500 nm.

FIG. 8 shows unstained TEM cross section of 4T1 derived CSC showing highcontrast at the outer and nuclear membrane (arrows) attributed to areasof high silica concentration.

FIG. 9 shows atomic force microscopy (AFM) images of the externalsurface of a calcined CSC derived from ASPC-1 cells.

FIG. 10 shows light microscopy and SEM images. Provided are (a) an imageof silicification of cells (AsPC-1) without chemical fixation; and (b)an SEM of calcined samples (AsPC-1, left panels; 4T1-particles, rightpanel) of unfixed cells. Arrows indicate areas of membrane rupture. Alsoprovided are (c) images showing silicification of fixed erythrocytesinduces cell lyses resulting in silica templated by erythrocytemembranes following calcination.

FIG. 11 is SEMs of 4T1 cells incubated in 0.5% Triton X-100 prior tosilicification, resulting in CSCs with altered surface morphologies andflattened regions.

FIG. 12 shows images of ASPC1 cells fluorescently stained for outermembrane (CellMask™ Orange) and cytoplasmic proteins (CellTracker™Green) before silicification (top panels), showing loss of membrane dyelocalization while the protein dye remained stationary.

FIG. 13 shows time lapse imaging of fixed AsPC-1 cells fluorescentlystained for outer membrane (CellMask™ Orange) under silicificationconditions (left panels) and methanol (right panels) both at 37° C.Scale bars: 10 μm.

FIG. 14 shows SEM images of gram-negative bacterial cells (E. coli)silicified using identical conditions to those of mammalian cells (100mM silicic acid, pH 3) indicates that following calcination (rightpanels) cellular-structure (left panels) is not stabilized (viaintra-cellular silicification) and thus obliterated followingcalcination (right panels). Scale bars: 2 μm.

FIG. 15 shows images showing the distribution of silica in CSCs, nuclearstaining, and lipid membrane reconstitution. Provided are (a) DIC andconfocal fluorescence images of AsPC-1 templated CSCs, showing thatsilica is continuous throughout the cytoplasm and nucleus as indicatedby PMPDO staining (middle panel). Right panel shows localization of DAPInuclear stain. Also provided are (b) a confocal fluorescence image sliceof substrate grown AsPC-1 CSCs showing surface localization of lipid andinternal location of the nucleus; and (c) an image of CSC particlessupporting lipid layers showing accumulation of esterase fluorogenicproducts, as indicated by the line labeled “CSC/lipid”. The line labeled“CSC/lipid (calc)” shows activity of calcined CSCs supporting lipidbilayers. Error bars describe the standard deviation (n 5) of themaximum intensity value. Scale bars: 10 μm, (a) and (b); 5 μm, (c).

FIG. 16 is a brightfield (left panel) and epifluorescence (middle andright panels) images of AsPC-1 cell/silica composite particles.

FIG. 17 shows a N₂ sorption isotherm of calcined CSCs templated from CHOcells.

FIG. 18 illustrates shape-preserving carbonization of 4T1 CSCs. Providedare (a) images showing pyrolysis of CSCs produces an opaque powdercomprised of particles that have retained cellular structure, shown in(b). Etching of the silica produces a carbon rich replica, as seen in(c). Also provided are (d) in situ electrical characterization ofcarbonized particles shows a 20 fold decrease in electrical resistanceacross a particle following silica etching. Scale bars for (b) and (c)panels, 2 μm; insets, 500 nm.

FIG. 19 shows representative TGA and DTA profiles for ca. 7 mg CHOderived CSCs (ramp rate, 10° C./min).

FIG. 20 shows SEM images. Provided are (a) images of a chicken embryoheart before and after silicification; and (b) images of a completechicken embryo before and after silicification.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional chemical synthetic methods and other biological techniqueswithin the skill of the art. Such techniques are well-known and areotherwise explained fully in the literature.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms in which case each carbon atomnumber falling within the range is provided), between the upper andlower limit of that range and any other stated or intervening value inthat stated range is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It is to be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow. It is understood that in the event a specific term is not definedherein below, that term shall have a meaning within its typical usewithin context by those of ordinary skill in the art.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein. Within its use incontext, the term generally refers to a single silicic acid compound orit's analog or derivative. In certain instances the term may also referto stereoisomers and/or optical isomers (including racemic mixtures) orenantiomerically enriched mixtures of disclosed compounds.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount or concentration of a compound or composition which,in context, is used to produce or affect an intended result.

The term “silicic acid” shall mean a family of chemical compoundscontaining the element silicon attached to oxide and hydroxyl groupswhich are capable of condensing and forming oligomeric and/or polymericsilicon dioxide coatings pursuant to the present invention. This familyof compounds includes at least one compound selected from the groupconsisting of orthosilicic acid (generally referred to as silicic acid),metasilicic acid, disilic acid and pyrosilicic acid, among others. Twosilicic acid derivatives which find use in the present invention includetetramethoxysilane (TMOS), tetraethoxysilane (TEOS) and mixtures thereofand are used as preferred silicic acid compounds according to thepresent invention because of their ready availability and the ease withwhich they may be used and controlled in solution. The silicic acidcompounds are generally used in a dilute, weak acid solution to providesilicone dioxide coatings onto structures which are found in thebiological materials which are exposed to the process according to thepresent invention.

The term “dehydration” is used to describe a simple process by which theCSCs which are produced are dehydrated to remove any excess water.Dehydration may be performed by simply adding a solvent such asmethanol, ethanol, or another volatile solvent is used to remove anyexcess water. In addition, the CSCs produced may be simply air dried atroom or slightly elevated temperature to remove water. Any process toremove water without impacting the structure of function of the CSCsproduced may be used in the dehydration step.

The term “reconstitution” shall refer to the ability of the CSCsaccording to the present invention to be reconstituted as biologicalmaterial from the silicone dioxide coated compositions. CSCs accordingto the invention are coated with polymeric silicone dioxide pursuant toexposure to silicic acid as disclosed herein. The silicic acid will forma polymeric coating of silicon dioxide on the biological surfaces of thematerial which are exposed to the silicic acid to produce CSCs. Uponexposure of the CSCs to solution (e.g., saline solution, bufferedsolution, a weak base solution or a dilute solution of HF, amongothers), the CSCs may be reconstituted back to their original biologicalstate.

The term “storage stable” shall mean storage for a period of at least afew days, preferably, at least several weeks, months or even years in aCSC form, which can be readily reconstituted in solution as describedabove,

The term “calcination” is used to describe a step to remove organicmaterials from the CSCs produced using the present method. Calcinationoccurs at elevated temperature (about 500-650° C. for a period ofseveral hours sufficient to remove organic material from the CSCs,resulting in a three dimensional structure of silicon dioxide whichremains after much of the biological material is removed from the CSC.

The present invention is directed to a generalized route to synthesizecell/silica composites (CSCs), analogous to diatom frustules, usingcells and other biological material including collections of cells,tissue, organs and related biological material as scaffolds directingcomplex structure formation. Inter- and intracellular heterogeneity fromthe nano- to macroscale is captured and dimensionally preserved in CSCsfollowing drying and high temperature processing allowing, for instance,size and shape preserving pyrolysis of cellular architectures to formconductive carbon replicas. The structural and behavioral malleabilityof the starting material provides vast opportunities to develop robustand economical biocomposites with programmed structures and functions.

In the present invention, a biological material is exposed to asilicification step comprising exposing the biological material tosilicic acid (or one or more of its derivatives and/or analogs such astetramethoxy silane and/or tetraethoxysilane at an effectiveconcentration (preferably ranging from about 50-250 mM, about 100 mM, oras otherwise described herein) in an aqueous solvent (including abuffered solvent), at a pH ranging from about 1.5-4.5 (preferably about3) in acid solution at low temperature (generally, less than about 0°C., preferably less than about 20° C., less than about 30° C., less thanabout 40° C. for a period sufficient (preferably, a few hours to about24 hours, preferably, about 8-15 hours) to produce cell/silicacomposition (CSC) material particles comprising primarily silicon,oxygen and carbon, optionally, dehydrating the CSC produced from thefirst step; and optionally calcinating the CSC particles at elevatedtemperature (generally, at about 500-650 C, preferably about 550-600 Cfor several hours, preferably about 3-4 hours, in air) to producecalcinated CSC. It is noted that the dehydration step and thecalcination are not required; rather the CSCs once formed, may be simplyremoved from the silicic acid, washed with solvent and used. Inaddition, the dehydration step is often not utilized when the CSCs whichare produced are subject to the calcination step—given that theconditions of calcination will tend to dehydrate the CSC particles.Accordingly, when CSC are not calcinated, for example, when it is founddesirable to maintain at least some of the organics in the CSCsproduced, a dehydration step is often used in the absence of acalcination step.

As an example of the present invention, chemically fixed mammalian cellswere incubated in dilute, silicic acid solutions as shown in FIG. 1.FIG. 1a is a schematic illustration of the process of cellsilicification. In a typical experiment, cells plated onto glasssubstrates were fixed using 2-4% fixative (formaldehyde andglutaraldehyde produced qualitatively similar results) for at least 10minutes. Cells were rinsed and immersed overnight (˜16 hrs) in asolution of 100 mM silicic acid at pH 3 and ˜40° C. resulting in acomposite comprising primarily silicon, oxygen, and carbon (cell/silicacomposites, CSCs). Calcination was performed in air at 550 to 600° C.for 3-4 hours which eliminated the majority of organics, as shown inFIG. 19. FIG. 1b shows brightfield images of the identical grouping ofdifferentiated AsPC-1 pancreatic carcinoma cells throughout the processshown in FIG. 1a : live, after fixation, silicification and drying, andcalcination. Insets show representative EDS spectra of cells at thevarious stages. Structural features and dimensions were observed at eachstage of the process to be nearly identical to those of the parent(cell) templates albeit with some minor cracking observed, from SEMimages of substrate bound, calcined CSCs, as shown in FIG. 1d .Additionally, features of hydrated living cells that were virtuallytransparent under brightfield microscopy appeared sharply resolved incalcined CSCs (e.g., the calcined sample imaged in FIG. 1c ) due to theincrease in refractive index contrast.

Cellular and sub-cellular morphology is dependent on genetic andenvironmental factors and therefore can be highly malleable andresponsive to, for instance, physical interactions with a substrate. Asshown in FIG. 1, the morphology of cells differentiated on a substratecan be faithfully captured in silica. Procedures were also developedunder conditions that give rise to more physically homogenous CSCparticles with high throughput. FIG. 2a is a schematic illustration ofthe formation of monodisperse CSC particles. Similar to silicificationas for adherent cells, CSC particles were derived by incubating cellsuspensions in TMOS on a shaker. For rinsing and drying, cells werepelleted and redispersed sequentially in rinse solutions (describedabove) and finally air-dried overnight from 100% methanol. Dehydrationresults in a dry powder comprised of monodisperse CSC particles. Asshown in FIG. 2b , cells fixed and silicified under suspensionconditions resulted in a population of essentially monodispersecomposite microparticles (e.g., average diameter of 4T1 derived CSCsshown was 8.9 μm±1.4) with complex surface features. The large panel inFIG. 2b shows a closeup SEM of a 4T1 templated CSC displaying a ruffledexternal surface. For fast growing CHO cells (doubling time ˜12 hours) astandard 225 cm² flask of adherent cells at 80% confluency (˜2.0×10⁷cells) yielded ˜10-20 mgs dry weight of CSCs, providing a means torapidly produce gram scale quantities from cell lines such as CHO usinglarge capacity bioreactors. See J. N. Warnock and M. Al-Rubeai,Biotechnol Appl Biochem 45, 1 (2006); and Z. Xing et al., BiotechnolBioeng 103(4), 733 (2009). This procedure was tested on cultured cellsderived from a variety of tissues. Similar particle sizes within a givenclonal cell line were observed but with widely differing surfacemorphologies both within and across the cell lines examined. FIG. 2cshows close-up SEMs of CSC particles derived from a variety of tissues.Insets show the whole particle. Membrane ruffles, filaments, blebs,clusters, and smooth surfaces—common features of cell membranedynamics—are captured in CSCs and calcined CSCs with high fidelity.Importantly, surface features of silica replicas can be directlymodified by inducing cell behaviors, such as apoptosis, and surfaceruffling prior to silicification. In particular, FIG. 3 shows SEM imagesof clusters of calcined (500° C., 3 hrs) silica replicas templated from4T1 cells incubated in 5 μm doxorubicin to induce apoptosis. The arrowsdenote apoptotic blebs and flementous surface structures that appear todegrade over the 150 min incubation. FIG. 2d shows calcined CSCstemplated from human erythrocytes showing normal to increasinglyabnormal/crenate morphology resulting from increasing levels of osmoticstress (left to right). FIG. 2e shows RBL-2H3 templated CSCs followingcalcination which display the predicted grainy to ruffled membranesurface transformation accompanying surface receptor crosslinking. SeeB. S. Wilson et al., Mol Biol Cell 9(6), 1465 (1998).

FIG. 4a shows an SEM of AsPC-1 templated CSC features prepared accordingto the present invention. FIG. 4b shows cells fixed and dehydrated usingstandard procedures. Magnified features are indicated by arrows in FIGS.4a and 4b . FIG. 4c shows SEM images of SK-OV-3 suspension culturedcells dried against a substrate with and without silicic acid treatment.External features of CSCs in FIG. 4a show more defined and detailedsurface structures compared to the identical cell line prepared usingthe well-established bench top electron microscopy preparationprocedures (i.e., no supercritical drying or rapid freezing) of fixationfollowed by careful dehydration in increasing concentrations of ethanoland drying from hexamethyldisilazane HMDS, shown in FIG. 4b , aprocedure shown to provide identical feature preservation as criticalpoint drying. See F. Braet et al., J Microsc 186, 84 (1997); and D. F.Bray et al., Microsc Res Tech 26(6), 489 (1993). Note thatsilicification can alter the size of nanoscale cellular features incomparison to drying from HMDS. Suspension cells silicified in solutionshowed particularly dramatic differences compared to non-silicifiedcells. As shown in FIG. 4c , CSC particles dried in contact with asubstrate (and even calcined) were resistant to deformation, remainingstiff and spherical, whereas the parent fixed cells deformedsignificantly with loss of surface features during drying, and of coursewere completely obliterated upon calcination in the absence of silicicacid treatment, as shown in FIG. 5. Thus, silicification acts tomechanically stabilize the cellular architecture during drying andparticularly during calcination, by forming a continuous, mechanicallyconnected interpenetrating network throughout the ‘cell hydrogel’,analogous to results from protein-templated silica hydrogels. See C. Y.Khripin et al., ACS Nano 5, 1401 (2011). The present invention cantherefore provide a simple alternative to common methods for specimenpreparation/preservation that does not require extensive optimization,expertise, or specialized equipment (e.g., critical point dryer), andparticularly when tolerance to extreme environments (e.g., temperature)is required.

FIG. 6 shows an SEM analysis of filopodia mean width of fixed cells (75nm), cell/silica composites (86 nm), and silica (79 nm) derived fromsubstrate-bound differentiated AsPC-1 cells, indicating a significantdifference in mean width (at 0.05 level using overall ANOVA, n >15 persample). The “cell” sample was prepared using EtOH:HMDS samplepreparation. Error bars indicate the standard error of the mean. SEMcomparisons of substrate bound differentiated AsPC-1 cells indicates anincrease in the size of nano-scale cellular features throughout theprocedure (˜10 nm increase in width of CSC filopodia outgrowths versusnon-silicified cells), which is attributed to silica deposition.

In order to examine the internal features of CSCs in greater detail,AsPC-1 cells were plated onto glass substrates, silicified, and dried.FIG. 7 shows cross-sectional imaging of CSCs enabled by a simplefracture technique. Glass substrates were scored on the surface oppositethe cells and fractured. Because of the brittle fracture characteristicsof the CSCs, cells lying across the fracture edge were often cleanlysectioned, allowing cross-sectional analysis using scanning electronmicroscopy. FIG. 7a shows a sectioned cell revealing intra-cellularstructures such as the nuclear membrane, indicated by 100 nm diameterring-like features (presumably nuclear pore complexes). The right panelis a close up view of the sectioned cell. Arrows indicate nuclear porecomplexes. FIG. 7b is an SEM section of a CSC showing multilayer,endoplasmic reticulum-like structures (arrows). FIG. 7c is an SEM of acalcined CSC sectioned on glass shows a 30 nm membrane templated silicastructure. FIG. 7d shows Filopodia-templated upright protrusions (1) areencased in a smooth silica membrane (2) overlying roughened,particle-based features (3) in a calcined and sectioned CSC. The arrowsin the insets of FIGS. 7b-7d point to the area of magnification.Comparison of fractured CSCs (e.g., FIGS. 7a and 7b ) and fracturedcalcined CSCs (FIGS. 7c and 7d ) showed no obvious difference in size orshape of internal features after calcination.

FIG. 8 shows unstained TEM cross section of 4T1 derived CSC showing highcontrast at the outer and nuclear membrane (arrows) attributed to areasof high silica concentration. Examining calcined structures, such asthose in FIGS. 7 c and 7 d, as well as TEM cross-sectional images of CSCparticles, such as those in FIG. 8, a conformal silica coating of ca. 30nm thick is apparent, elaborated around filapodia-like features (shownin FIG. 7d ) and encasing the intracellular-templated structures andvoid spaces. In a eukaryotic cell, the membrane is defined by thephospholipid bilayer anchored to the cell cortex via membrane boundproteins. The cortex is composed of fibrous proteins such as spectrinand actin, forming a meshwork that provides mechanical strength to themembrane. High resolution atomic force microscopy (AFM) imaging ofrelatively flat regions of calcined external surfaces were featurelessat ˜2 nm resolution indicating the absence of a primary feature orparticle size. FIG. 9 shows AFM images of the external surface of acalcined CSC derived from ASPC-1 cells. Analysis of the height image(right panel; scanned area of the box in the left panel) was used tomeasure surface roughness (standard deviation, σ=1 nm) within errorattributed to the tip radius (<2 nm). Similar observations were made inAFM studies of select diatom cell surfaces. See M. Hildebrand and M. J.Doktycz, Pflugers Arch 456, 127 (2008). In comparison, silica templatedby single component protein hydrogel scaffolds was observed to begranular with a primary feature size of ˜16 nm. See C. Y. Khripin etal., ACS Nano 5, 1401 (2011).

A series of experiments were conducted to understand the mechanism ofcell silicification. First, cells subjected to silicification conditionswithout fixation were observed to swell significantly, as a result ofhypotonic stress, but nonetheless formed CSCs (albeit with drasticdifferences in morphology due to membrane swelling and other stressesincurred during silicic acid incubation. Therefore, fixation can enablepreservation of shape in the natural state, but is not required for allapplications. FIG. 10a is an image of silicification of cells (AsPC-1)without chemical fixation, resulting in cell swelling indicative ofhypo-osmotic cellular stress. FIG. 10b is an SEM of calcined samples(AsPC-1, left panels; 4T1-particles, right panel) showing the resultantcell templated silica with altered morphology. The light arrows indicateareas of membrane rupture. Erythrocytes are particularly sensitive toosmolarity and were found to lyse in the silicic acid solution whenfixed for short time scales. FIG. 10c shows that silicification of shorttime fixed erythrocytes induces cell lyses resulting in silica templatedby erythrocyte membranes following calcination. Through a modifiedfixation process and use of an osmotically balanced silicic acidsolution (addition of 0.9% NaCl), CSCs and calcined erythrocytes silicareplicas were achieved that faithfully replicated the parent cellmorphology shown in FIG. 2 d.

As shown in FIG. 11, complete solubilization of the membrane of fixedcells using a mild detergent (0.5% Triton X-100) prior to silicificationresulted in CSCs with deformed features, most likely incurred as aresult of settling against the reaction tube surface. However, stainingof the outer lipid membrane and intracellular proteins followed bysilicification showed some de-localization of lipid following incubationin the silicic acid solution (also, confirmed by post-staining CSCsusing a lipid-associating dye) while the protein dye remainedstationary. FIG. 12 shows ASPC1 cells fluorescently stained for outermembrane (CellMask™ Orange) and cytoplasmic proteins (CellTracker™Green) before silicification (top panels) showing loss of membrane dyelocalization while the protein dye remained stationary. Membranestaining of cells following silicification produced qualitativelysimilar results. Triton X-100 is not expected to disrupt the corticallayer or other cytoskeletal constituents, or denature most proteins atthis concentration. Taken together, these results indicate that thewhole membrane complex (lipid bilayer+cortex) is necessary to maintainthe mechanical integrity of CSC surfaces, but that a portion of thelipid component is gradually displaced during silica deposition.

Indeed, time-lapse imaging of a lipid membrane dye indicates that thepresence of dilute methanol (hydrolyis product of TMOS) in thesilicification solution provides relatively slow and mildpermeabilization of cell membranes (compared to methods used forimmunostaining, such as Triton and 100% methanol) that enables silicaprecursors to penetrate into the cell while maintaining the mechanicalintegrity of external cell features during silica deposition. FIG. 13shows time lapse imaging of fixed AsPC-1 cells fluorescently stained forouter membrane (CellMask™ Orange) under silicification conditions (leftpanels) and methanol (right panels) both at 37° C. Delocalization offluorescent dye from the cell exteriors with concurrent increase ininterior fluorescence indicates that the timescale for membranepermeabilization varies from cell to cell, occurring over minutes tohours (arrows in left panels). Similar observations in 0.4 methanol(arrows in right panels) indicate membrane permeabilization is primarilydue to incubation in methanol generated from the acid catalyzedhydrolysis of the silicic acid precursor TMOS.

Additionally, CSCs derived from E. coli do not retain cellular structurefollowing calcination which indicates incomplete silica templating, mostlikely as a consequence of inhibited intracellular penetration of silicaprecursors past the prokaryotic cell envelope. FIG. 14 shows SEM imagesof gram-negative bacterial cells (E. coli) silicified using identicalconditions to those of mammalian cells (100 mM silicic acid, pH 3)indicates that following calcination (right panels) cellular-structure(left panels) is not stabilized (via intra-cellular silicification) andthus obliterated following calcination (right panels).

Silica localization throughout the CSC was observed duringsilicification using PDMPO:([2-(4-pyridyl)-5-((4-(2-dimethylaminoethylamino-carbamoyl)methoxy)phenyl)oxazole]),which has been shown to incorporate with silica as it condenses. See B.Tesson and M. Hildebrand, PloS one 5, e14300 (2010). FIG. 15 shows thedistribution of silica in CSCs, nuclear staining, and lipid membranereconstitution. FIG. 15a shows DIC and confocal fluorescence images ofAsPC-1 templated CSCs showing that silica is continuous throughout thecytoplasm and nucleus following incubation for 16 hours, as indicated byPMPDO staining (middle panel). The right panel shows localization ofDAPI nuclear stain. This indicates that although silica condensation islikely to occur over variable timescales at the (macro)molecular scale,it eventually infiltrates all discernible subcellular structures andorganelles—with the notable exception of large, fluid filled vacuoles.FIG. 16 shows a brightfield (left panel) and epifluorescence (middle andright panels) images of AsPC-1 cell/silica shows silica localization(PDMPO panel) throughout the cellular interior—including the nucleus(DAPI panel)—with the noticeable exception of vacuole-type structures(arrows in left and middle panels). Further, the nuclear stain4′,6-diamidino-2-phenylindole (DAPI) is shown to localize exclusivelywithin the nuclear region of CSCs with little background signal, asshown in FIGS. 15a and 15b . This indicates that when CSCs are incubatedin an aqueous solution of the dye molecule, the DNA helical structureremains intact and molecularly accessible within the nucleus—despitesilicification throughout the nuclear region.

N₂ sorption isotherms obtained from CHO-templated silica particles(representing a silica imprint of the internal and external cellularstructure) indicated a BET surface area of ˜365 m²/g and a broad rangeof pore dimensions, although with no appreciable microporosity. FIG. 17shows a N₂ sorption isotherm of calcined CSCs templated from CHO cells.The lack of a distinct condensation step in the adsorption branchindicates a wide pore size distribution (PSD); a fit to the adsorptionbranch using a hybrid DFT model for cylindrical pores in silica (topinset) shows that the material contains a broad range of poredimensions, although with no microporosity (pore size less than 2 nm).See M. Jaroniec et al., A new method for the accurate pore size analysisof MCM-41 and other silica based mesoporous materials, FifthInternational Symposium on the Characterization of Porous Solids, COPSV, Heidelburg; Unger, K. K.; Kreysa, G.; Baselt, J. P., Eds. Elsevier:Heidelburg, pp 71-80 (1999). Because there is no plateau in theadsorption branch at high P/P₀, the total porosity for pores greaterthan ca. 40 nm cannot be determined from this isotherm. However,hysteresis in the desorption branch—likely due to a bottleneck structurewithin a pore network—contains two inflection points (derivativeincluded as bottom inset) at P/P₀=0.46 and 0.87, which is indicative oftwo populations of internal porosity. The two populations of mesoporerestrictions suggests the presence of large interstitial pores definedby the volume between cellular structures connected through two subsetsof smaller pores.

The results from the above series of experiments indicate that thesilica deposition process occurs throughout the complete volume of thecell to produce a faithful replica of the exterior and interior cellularstructures. Based on the featurelessness of silica deposits in selectareas, it can be concluded that deposition at pH 3 involves weaklycharged monomeric or small oligomeric silicic acid species that interactnon-covalently with the crowded biomolecular components comprising thecell. The high fidelity replication and self-limiting characteristicssuggest a mechanism where silicic acid is distributed uniformly over andthroughout the cell scaffold where it undergoes acid or base catalyzedcondensation promoted by the spectrum of proximal functional groups suchas protein surface residues. In this manner, the process is inherentlyself-limiting to form a continuous silica replica throughout the cell.Remarkable is that the silicified cell, although nanostructured,withstands drying and sintering to 550° C. with minimal shrinkage, asshown in FIG. 6. Generally, drying (capillary) and sintering stresseswould result in enormous volumetric changes. See C. J. Brinker and G. W.Scherer, Sol-gel science (Academic Press, San Diego) (1990). The absenceof appreciable shrinkage speaks to the mechanical integrity of thecell-catalyzed silica replica. The absence of primary particles andmicroporosity reduces greatly both drying and sintering stresses, whichscale roughly inversely with particle or pore size. One mechanistichypothesis consistent with these observations is that at pH 3 wheresilicic acid monomers and oligomers are uncharged, silicic acidincorporates within the continuous hydrogen bonded water networkencompassing cellular surfaces where it becomes locally concentrated andsubsequently condensed amphoterically via surface moieties (e.g. acidicand basic protein residues). See T. Coradin et al., Colloids Surf B 29,189 (2003); and R. K. Iler, The chemistry of silica: solubility,polymerization, colloid and surface properties, and biochemistry (Wiley,New York) (1979).

In essence, the structural complexity of cells is captured viaself-limiting nanoscale replication in a hard material, providing aplatform in which to preserve and reconstitute cellular functions. Forexample, amphiphilic lipid bilayers introduced as liposomes localize(selectively as compared to on the adjoining substrate) on the outersurfaces of CSCs demonstrating that the membrane lipid component could,in principle, be reconstituted. Subsequent, incubation with a lipiddiffusible fluorogenic stain used to assess cellular viability indicatedretention of some level of enzyme activity; sequestration of the dye(based on esterase cleavage to form a lipid insoluble fluorophore) wasobserved in CSCs supporting lipid membranes versus calcined CSCs, asshown in FIG. 15c . FIG. 15c is an image of CSC particles supportinglipid layers showing accumulation of esterase fluorogenic products, asindicated by the line labeled “CSC/lipid”. The line labeled “CSC/lipid(calc)” shows activity of calcined CSCs supporting lipid bilayers. Theseinitial results provide an avenue to begin to explore CSCs as analternative route to biocatalyst stabilization where the currentstate-of-the-art employs pre-fabricated (mesoporous) silicas forsubsequent enzyme loading. See U. Hanefeld et al., Chem Soc Rev 38, 453(2008); S. Hudson et al., Angew Chem Int Ed 47, 8582 (2008); L. Betancorand H. R. Luckarift, Trends Biotechnol 26, 566 (2008); and D. Avnir etal., J Mater Chem 16, 1013 (2005). By using this general approach as astarting point, more complex and specific biocatalyst stabilization canbe targeted, by stabilizing enzymes and enzyme complexes in theiroptimized, crowded in vivo configurations.

Finally, the ability to replicate both surface and intracellularmolecular architectures with silica provides opportunities toinvestigate shape-preserving chemical transformations of CSCs to othermaterials, for instance, using approaches analogous to those developedfor diatom silica. See K. H. Sandhage et al., Handbook ofBiomineralization: Biomimetic and bioinspired chemistry, 235 (2007); D.Losic et al., Adv Mater 21, 2947 (2009); and Z. Bao et al., Nature 446,172 (2007). Therefore, the ability of CSCs to render porous carbonstructures, a class of materials with substantial utility in fuel cell,decontamination, and sensor technologies, was investigated. FIG. 18illustrates the shape-preserving carbonization of 4T1 CSCs. FIG. 18ashows the pyrolysis of CSCs produces an opaque powder comprised ofparticles that have retained cellular structure, as shown in FIG. 18c .Etching of the silica produces a carbon rich replica. FIG. 18d shows insitu electrical characterization of carbonized particles shows a 20 folddecrease in electrical resistance across a particle following silicaetching. The CSC particles were subjected to high-temperature pyrolysisconditions (900° C., 4 hrs, under N₂ atmosphere) which resulted in anopaque powder, as shown in FIG. 18a , with individual particles(carbonized-cell/silica composites, c-CSCs) displaying similarmorphologies to that of the starting material, as shown in FIG. 18b .Subsequent dissolution of the silica support (6 M potassium hydroxide(KOH), 4 days) resulted in free-standing carbon particles retainingcellular morphologies, as shown in FIG. 18c . In situ SEM electricalcharacterization, as shown in FIG. 18d , showed ohmic conductivitythrough the particles. Representative IV curves for c-CSCs and carbonreplicas are shown in the lower panel of FIG. 18d . Note that removal ofthe insulative silica support decreased particle resistance ˜20 fold.These results indicate that the wide heterogeneity of in vitro softcellular architectures can now be considered for use as a feedstock formost materials processing procedures, including those requiring hightemperature and pressure.

Therefore, the present invention provides a simple method to derivefunctional biomorphic composites, silica “frustules”, and carbonreplicas from cells, which can allow straightforward customization ofstructure and function via chemical and genetic engineering. This methoddoes not require pre-infiltration of templating molecules (e.g.,cationic polymers) or multistep layer by layer assembly and is distinctfrom other inorganic biotemplating strategies that simply coat externalsurfaces to produce hollow shells or low fidelity inverse structuresfollowing calcination. See L. Niu et al., Angew Chem Int Ed 50, 11688(2011); O. Paris et al., MRS Bull 35, 219 (2010); and M. Dickerson etal., Chem Rev 108, 4935 (2008). In contrast to the majority of studiesdescribing cell encapsulation in silica where the primary goal ofmaintaining cell viability necessitates reaction conditions near neutralpH and cells become physically entrapped within (non-conformal) gels,with the method of the present invention the charge of silicic acid isessentially neutral (pH 3) and thus hydrogen bonding and othernon-covalent silica/molecular interactions govern deposition. See C. F.Meunier et al., J. Colloid Interface Sci. 342(2), 211 (2010); T. Coradinet al., Colloids Surf B 29, 189 (2003); R. K. Iler, The chemistry ofsilica: solubility, polymerization, colloid and surface properties, andbiochemistry (Wiley, New York) (1979); and C. Y. Khripin et al., ACSNano 5, 1401 (2011). To date, individual cellular/biomolecularcomponents, peptides, proteins, lipid vesicles, polysaccharides,cytoskeletal filaments, etc. have all been shown to interact with, andoften template silica in vitro but with no control over 3D structure.See O. Paris et al., MRS Bull 35, 219 (2010); A. Bassindale et al., JMater Chem 19, 7606 (2009); M. Dickerson et al., Chem Rev 108, 4935(2008); and R. K. Iler, The chemistry of silica: solubility,polymerization, colloid and surface properties, and biochemistry (Wiley,New York) (1979). Presented on and within a cell, these collectivesilica/molecular interactions are exploited in the present method undermolecularly crowded environments using stable sols (e.g., limitedhomo-polymerization, no gel formation, etc.) such that deposition istargeted to cell structures, resulting in a process that is inherentlyconformal and self-limiting due to slow solution silica polymerizationkinetics. See R. K. Iler, The chemistry of silica: solubility,polymerization, colloid and surface properties, and biochemistry (Wiley,New York) (1979). The generalizability of this method can enable thesynthetic production of complex and durable composites and minerals withstructural diversity approaching that of natural biomineralizingmicroorganisms.

Using the methods described above for single cells, the presentinvention can be used preservation/replication of higher order animalstructures such as soft tissue and whole organisms. FIG. 20A shows achicken embryo heart (box outline is further magnified in right panel).FIG. 20B shows a complete chicken embryo. Silicification takes placeover the course of 3-8 days but remarkably, the entire architecturalhierarchy of macro-scale features down again to single cells andsub-cellular structures is replicated in silica following calcination at500° C. for 6-12 hrs, as shown in the right-hand panels of FIGS. 20A and20B.

The present invention has been described as a method to synthesizecell/silica composites. It will be understood that the above descriptionis merely illustrative of the applications of the principles of thepresent invention, the scope of which is to be determined by the claimsviewed in light of the specification. Other variants and modificationsof the invention will be apparent to those of skill in the art.

We claim:
 1. A method to synthesize cell/silica composites (CSCs),comprising incubating a plurality of cells in a silicic acid solution toprovide cell/silica composite particles.
 2. The method of claim 1,wherein the plurality of cells comprises mammalian cells, prokaryoticcells, plant cells, cultured cells, tissue, or biological organism. 3.The method of claim 1, wherein the plurality of cells is genetically,chemically, or physically modified.
 4. The method of claim 1, furthercomprising fixing the plurality of cells in a fixative prior to theincubating step.
 5. The method of claim 1, wherein the silicic acidsolution is osmotically balanced.
 6. The method of claim 1, furthercomprising plating the plurality of cells on a substrate prior to theincubating step.
 7. The method of claim 1, further comprising patterningthe plurality of cells on a surface prior to the incubating step.
 8. Themethod of claim 1, wherein the plurality of cells are suspended in thesilicic acid solution.
 9. The method of claim 8, further comprisingdehydrating the cell/silica composite particles to provide monodispersecell/silica composite particles.
 10. The method of claim 1, wherein thesilicic acid solution comprises tetramethoxysilane, tetraethoxysilane,or a mixture thereof.
 11. The method of claim 1, further comprisingcalcinating the cell/silica composite particles at an elevatedtemperature to provide silica replicas.
 12. The method of claim 11,wherein the elevated temperature is greater than 500° C.
 13. The methodof claim 11 further comprising reconstituting a cellular function on thesilica replicas.
 14. The method of claim 1, further comprisingpyrolyzing the cell-silica composite particles at a high temperature inan inert atmosphere to provide carbonized-cell/silica compositeparticles.
 15. A population of CSCs which are produced by a methodaccording to claim
 1. 16. The population of CSCs according to claim 15which is storage stable.
 17. A cell/silica composite (CSC) comprising atransformed cell, which further comprises a silicon dioxide coating ontoa structure which is found in said cell.
 18. The CSC of claim 17,wherein the coating is a conformal coating.
 19. The CSC of claim 17,further comprising a preserved shape and/or feature, as compared to theoriginal cell.
 20. The CSC of claim 17, wherein the CSC is dehydratedand/or calcined.